The ability to specifically recognize, control and eliminate infections and cancer is one of the hallmarks of human immunity. The immune system can be partitioned into 1) the non-specific ‘innate’ system, with responses mediated by macrophages, dendritic cells, natural killer cells and neutrophils and recognizing a relatively small number of pathogen-associated molecular patterns, and 2) the highly specific CD4+ and CD8+ T cell-mediated ‘adaptive’ immune system, potentially recognizing millions of different peptide antigens.
Recognition of specific antigens by CD8+ T cells of the adaptive immune system is mediated by highly diverse T cell receptors (TcR). T cells bearing a single TcR can recognize a specific peptide antigen presented by an appropriate MHC molecule, resulting in an ‘adaptive’ immune response with specificity for the presented peptide antigen. The CD8+ T cell ‘adaptive’ immune response to ‘foreign’ antigens is well characterized in viral infection, but CD8+ T cells specific for mutated or non-mutated ‘self’ antigens may be found in other conditions such as cancer and autoimmune disease (1-7).
In acute viral infection, virus-derived antigens are processed and presented by antigen presenting cells (APC) to naïve T cells that express TcRs capable of recognizing the viral antigen and in humans are characterized by cell surface expression of CD45RA and CD62L and absence of CD95 expression. The activated antigen-specific naïve T cells then rapidly proliferate and differentiate into an effector T cell population. The vast majority of effector T cells subsequently die, but a small fraction survives and become memory T cells (8, 9). On re-challenge with the virus, the surviving memory T cell population has the capacity to rapidly proliferate and differentiate into an effector population to rapidly contain the infection and protect the host. Antigen-specific CD8+ T cell memory has been described to persist up to 75 years, even in the absence of antigen rechallenge—essentially providing immunity to that antigen for the lifetime of the host (10).
The surviving memory T cell population is highly heterogeneous and comprises three main subsets in humans termed central memory (CM), effector memory (EM) and effector memory RA+ (EMRA). These subsets, and sub-populations thereof, differ in phenotype, ontogeny, homing, proliferative capacity and cytokine secretion, and might have distinct roles in maintenance of immune memory (11-13). The distribution of memory subsets can be affected by variation in conditions at priming such as the nature of the APC and antigen, the antigen density and the presence of cytokines, costimulatory molecules and inflammatory mediators (8). Once established, CD8+ T cell memory can persist in the absence of antigen (14, 15). Memory CD8+ T cell populations undergo homeostatic (steady state) proliferation and different subsets appear to have different rates of turnover in vivo (16). Interleukin-(IL−) 15 is a critical mediator of homeostatic proliferation and IL-7 is important for the survival of established memory responses (17-22). Despite advances in our understanding of the acute effector response to viral infection and the transition to a stable memory response, the mechanisms by which CD8+ memory is established and maintained have not been elucidated.
It has been hypothesized that a population of ‘stem-cell’ like T cells with the capacity to self-renew and differentiate into effectors may provide for the maintenance of immunologic memory (23). A putative memory stem cell was recently identified in a murine model of graft versus host disease (GVHD) (24). After secondary transfer from mice with GVHD, only post-mitotic CD8+/CD44lo/CD62Lhi/Sca-1hi memory cells were able to initiate GVHD, give rise to memory (CM and EM) and effector subsets, and retain replicative potential. Another study in mice demonstrated asymmetric cell division, a characteristic stem cell self-renewal mechanism, after the first encounter of naïve T cells with antigen (25). After the first division, the progeny ‘distal’ and ‘proximal’ to the immunologic synapse were programmed for a memory and effector phenotype, respectively. These studies suggest that antigen-specific CD8+ T cell memory may be maintained by a long-lived population with stem cell features and the capacity to self-renew. To date, no candidate population has been identified in the human.
The identification of the phenotype of long-lived memory CD8+ T cells or memory stem cells will have profound implications for investigation and therapy of infections, cancer and autoimmune diseases. We have used multiparameter flow cytometry to identify memory CD8+ T cell populations in humans with features consistent with stem cell behavior and long survival. A characteristic of hematopoietic and cancer stem cells is the ability to efflux chemotherapy drugs and fluorescent dyes (26-30). We found that subpopulations of CM and EM CD8+ T cells also had the capacity to rapidly efflux fluorescent dyes and chemotherapy drugs and we hypothesized that these cells could be responsible for the observed chemoresistance of CD8+ T cell memory after severely myelosuppressive chemotherapy. In vitro studies demonstrated that CM and EM subsets with the capacity to rapidly efflux rhodamine 123 (Rh123) (referred to as CMhi and EMhi, respectively for high efflux capacity) were more resistant to apoptosis than their non-effluxing counterparts in response to cytotoxic chemotherapy and that chemoresistance was attenuated by blockade of ATP-binding cassette cotransporter efflux channels.
Gene expression profiling studies show that CMhi and EMhi CD8+ T cells comprise similar, yet distinct subsets. In addition, they have gene expression profiles that are unique and distinct from those of other memory or naïve CD8+ T cell populations. Further studies showed that the immunophenotype of Rh123 effluxing memory populations was similar to previously described ‘memory stem cells’ in mice and the ‘distal pole-derived memory cells’ after asymmetric division of naïve murine CD8+ T cells. CMhi and EMhi populations harbor CD8+ T cells expressing a polyclonal TcR repertoire and CMV, EBV and influenza antigen-specific CD8+ T cells can be identified within the subsets.
CMhi and EMhi CD8+ T cells are refractory to polyclonal stimulation with OKT3, demonstrating reduced proliferation and cytokine secretion, compared to non-effluxing CD8+ T cells. They also exhibit low intracellular calcium flux in response to ionomycin stimulation. The reduced proliferation and cytokine secretion can be partially rescued with costimulation and inflammatory cytokines. Despite the reduction in secretion of many inflammatory cytokines, CMhi and EMhi secreted IL-17 in response to PMA-ionomycin stimulation in contrast to other memory CD8+ T cell subsets.
The refractory nature of CMhi and EMhi may allow them to remain in a quiescent state, avoiding differentiation in response to antigenic stimulation in all but the most inflammatory conditions. The observations that CMhi show high 3H-thymidine uptake and CFSE dilution in response to the homeostatic cytokines, IL-7 and IL-15, suggests that these chemoresistant cells may proliferate during the lymphocyte nadir after myelosuppressive chemotherapy when IL-7 and IL-15 levels are elevated, and potentially repopulate the memory CD8+ T cell compartment.
CMhi and EMhi are found in very low numbers in cord blood. They are found in high numbers in early adulthood and decline with advancing age. A population that arises after antigen exposure in early life and is gradually exhausted with repeated inflammatory antigenic stimuli in adulthood would be consistent with a putative memory stem cell. It would also be consistent with recognized decreased efficacy of vaccination in elderly subjects.
Identification of CMhi and EMhi can be easily achieved in vitro using Rh123 efflux assays; however the use of Rh123 in functional studies or clinical grade isolation is problematic. Therefore, we used multiparameter flow cytometry to search for cell surface markers that might distinguish this subset of cells in human blood samples. We found that high expression of CD161 and/or IL-18Rα identifies subsets that are enriched in CMhi and EMhi, facilitating identification and isolation of these cells in the absence of in vitro culture or exposure of the cells to Rh123 toxicity.
There is extensive evidence suggesting that memory CD8+ T cells have a role in prevention, control and therapy of infections, cancer and autoimmune diseases (1-7). Clinical studies of CD8+ T cell adoptive transfer in stage IV melanoma resulted in up to 51% CR/PR and demonstrated that persistence of the transferred tumor-specific T cells was critical for efficacy (31, 32). Findings in CMV-specific adoptive transfer studies also demonstrated the need for persistence, strongly supporting the hypothesis that the establishment of long-lived memory responses may be essential for successful control and protection against tumors and infection by adoptive T cell transfer (33). These studies are complemented by work in mice, demonstrating the critical role of persistent memory CD8+ T cells in eliminating clinically apparent cancer and establishing healthy equilibrium in occult cancer (4).
Despite the evidence that perturbations in memory CD8+ T cells are important in disease processes, attempts to specifically ablate, augment or transfer antigen-specific immune memory have met with limited success. Clinical responses after transfer of CD8+ lines or clones have been shown previously, but have been sporadic, related to their unpredictable persistence in vivo (32). Recent studies in non-human primates have shown CM-derived CD8+ clones can persist for up to one year after infusion, whereas EM-derived clones die rapidly by apoptosis, despite the fact that both populations shared an effector phenotype prior to transfer (34) This suggests that effector CD8+ T cells may retain an intrinsic program, derived from their cell of origin, which determines their survival in vivo after antigenic stimulation and clonal expansion. The implication is that clones or lines must be generated from appropriately programmed subsets if transferred memory CD8+ T cells are to persist in vivo. CMhi and EMhi are subsets with characteristics that suggest they are programmed for long survival.
The identification of memory CD8+ T cell subsets with appropriate programming for persistence will facilitate transfer of CD8+ T cell-mediated immunity against specific antigens. CD8+ T cells with programming for persistence and long-lived survival may also be useful as delivery vehicles for therapeutic genes. In addition to being amenable to long-term survival for maintenance of therapeutic immunity or gene delivery after adoptive transfer, CMhi and EMhi long-lived memory cells may act as targets for immunosuppression by ablation of antigen-specific memory responses through the use of toxin-conjugated CD161 and/or IL-18Rα monoclonal antibodies. This form of therapy may have a role in autoimmune diseases and graft versus host disease.